1. Field of the Invention
The present invention relates to a selective permeable membrane and an electrode using it to be favorably employed, for example, in an immobilized biocatalytic electrode in a system for detecting the formation or consumption of a substance which is detectable electrochemically.
2. Description of the Prior Art
The prior art relating to the selective permeable membrane is described below together with the background technology of its application field.
Recently, thanks to the advancement in biochemistry and other studies, the hitherto unknown bioreactions are gradually disclosed to be applied in the fields of chemical industries.
The scope of the biochemical reactions catalyzed by cells and enzymes is broad, and in particular attention is focused on the so-called biosensing field which is intended to apply the biochemical reaction as the means for detection of substances. By the use of biosensing techniques, the hitherto immeasurable substances or substances which took too much time and labor to measure can now be measured easily, and it is being practically employed in the fields of environmental measurement, food analysis, and medical analysis.
Generally, the biocatalysts such as enzymes involve many advantages including:
1. High specificity of reaction.
2. Capability of measurement in mild conditions.
3. High sensitivity to measure trace substances.
By contrast, since biological materials are used as the catalysts, the following disadvantages are also known.
1. Biocatalysts such as enzymes are expensive.
2. Reaction conditions such as temperature and pH are limited.
3. The catalytic function of biocatalysts such as enzymes is lost by various factors, that is, inactivation occurs.
To solve such problems, it is proposed to immobilize the biocatalysts, and many methods have been presented so far. Among them, the method of immobilizing the biocatalyst by using a covalent bonding crosslinking agent by crosslinking reaction is preferred for practical use because its bond is firm as compared with adsorption or ion bonding.
When applying such immobilized biocatalyst in measurement of a substance, a biocatalyst is used in combination with the detection means for detecting a substance formation or consumption depending on its biochemical reaction. As such detection means, an electrochemical detector, a fluorescent detector, a thermal detector, or the like may be applied. In particular, the electrochemical detecting method of consumption of oxygen or formation of hydrogen peroxide in biochemical reaction by an immobilized enzyme is most widely employed because of the simplicity of apparatus compositon, high sensitivity of detection, and speed of reaction rate.
However, when the hydrogen peroxide electrode is used, not only the hydrogen peroxide produced by biochemical reaction, but also undesired substances such as ascorbic acid contained in the sample are detected, and the reliability of measurement is known to be lowered. To solve this problem, it is attempted to place a selective permeable membrane between the immobilized enzyme membrane and the hydrogen peroxide electrode. That is, near the surface of the hydrogen peroxide electrode, a selective permeable membrane for limiting permeation of substances other than the object substance and an immobilized enzyme membrane are disposed in this order through a buffer solution or the like, or a selective permeable membrane and an immobilized enzyme membrane are directly formed in this order on the electrode surface. As a result, detection of undesired substances may be prevented, while the precision of measurement may be enhanced.
Conventionally as such a selective permeable membrane, for example, acetyl cellulose was used as disclosed in the Japanese Laid-open Patent No. 60-56254. To manufacture such selective permeable membrane, however, it is necessary to use an organic solvent that is hard to handle. Moreover, in order to obtain a desired selective permeability, the drying time and other conditions must be adjusted delicately, which requires an enormous amount of time and a complicated operation.
The present inventors have previously proposed a method of forming a selective permeable membrane easily by using protein and crosslinking agent (the Japanese Laid-open Patent No. 63-182559). However, not limited to selective permeable membranes, generally, the immobilized protein membranes formed by using protein and crosslinking agent are low in physical strength, and are hard to handle, and the durability is inferior.
The problem of such physical strength of immobilized protein membrane cannot be radically solved even if the protein is immobilized on a carrier having a high physical strength, for example, because the immobilized protein membrane itself is brittle and may be partly exfoliated.
Thus, the problems of the selective permeable membrane still remain unsolved at the present without proposal of any effective solutions. Generally, while not directed to a selective permeable membrane, the method of enhancing the strength of immobilized protein membrane formed by immobilizing the protein which is a biocatalyst such as enzyme is disclosed, for example, in the Japanese Patent Publication No. 58-49821. In this method, the enzyme as biocatalyst is immobilized together with amino and imino high polymers, and the physical strength of the enzyme immobilized membrane is enhanced. When this method is applied in the selective permeable membrane, however, the properties of immobilized protein may be changed, and it is difficult to obtain a desired selective permeability.
Futhermore, as disclosed in the Japanse Laid-open Patent No. 62-32352, a method of reinforcing the strength of enzyme immobilized membrane by immobilizing the enzyme which is a protein on a polyester cloth is proposed. In this method, however, the thickness of the protein membrane increases, and when applied in formation of selective permeable membrane, the measuring sensitivity and speed of the immobilized enzyme electrode are worsened.
Hereinbelow is a description of the prior art relating to immobilized enzyme membrane together with the background technology of its application field.
The methods for immobilizing enzyme may be roughly classified as follows:
1. The absorbing method to immobilize the enzyme by physically absorbing it on a water-insoluble carrier.
2. The crosslinking method to make the enzyme insoluble by a crosslinking reaction between the enzyme and another enzyme or protein by using a reagent having multi functional groups.
3. The entrapping method to wrap the enzyme in a fine lattice of high molecular gel or to cover it with a capsule of semipermeable high molecular membrane.
Among these methods, the crosslinking method is relatively widely used because the enzyme is firmly immobilized and the operation for immobilizing the enzyme is relatively easy. In particular, aldehydes as represented by glutaraldehyde are high in reactivity with enzyme, and the enzyme can be immobilized in a relatively moderate conditions, and hence they are used widely.
In the immobilizing method of an enzyme using aldehydes, for example, a mixed solution of the enzyme to be immobilized and aldehyde is prepared in advance. Next, this mixed solution is applied on the carrier or this carrier is dipped in the mixed solution (immersion method), and an immobilized enzyme membrane is formed. In the immersion method, however, it is difficult to control accurately the amount of the solution on the carrier surface, and hence it is difficult to control the immobilizing amount of enzyme precisely.
On the other hand, when a specific volume of mixed solution is applied on the carrier in order to immobilize the enzyme with high reproducibility, the enzyme and its crosslinking reagent of aldehyde are mixed preliminarily, and the crosslinking reaction is gradually progressed in the mixed solution. Therefore, in the case of continuous mass production of a multiplicity of immobilized enzyme, the mixed solution may be immobilized in the midst of production process. For example, as proposed in the Japanese Laid-open Patent No. 61-245051, in the case of formation of a quantity of immobilized protein membranes by continuously ejecting a mixed solution of protein and aldehyde from the nozzle, the viscosity of the mixed solution of protein and aldehyde is gradually raised to be finally immobilized, and therefore it is difficult to form the immobilized protein membranes continuously in some degree.
Therefore, in order to immobilize the enzyme homogeneously with high reproducibility, it is desired to prepare preliminarily a mixed solution of enzyme and aldehyde at specific ratio, apply it on the carrier, and start crosslinking reaction. In this method, the excellent enzyme electrode is obtained, however, there is the problem of lowering of the working efficiency due to increase of viscosity and hardening of the mixed solution in the manufacturing process.